Marijuana (Cannabis sativa) has been known for many centuries as a psychoactive agent and also for its medicinal properties. In humans, it has been used as an analgesic, anti-inflammatory, immunosuppressant and anticonvulsant. Recently, two cannabinoid receptors have been cloned, the central and the peripheral cannabinoid receptors. The central cannabinoid receptor (CB1) was first found in the brain, but also occurs in the testis. The peripheral cannabinoid receptor (CB2) seems to be predominantly expressed in the spleen. This may explain the immunosuppressive properties of marijuana and its active component, delta-9-tetrahydrocannabinol (THC). Previous work has suggested that CB2 is present in the marginal zone of the spleen, but there has been discrepancy as to its cellular localization. We used specific immunocytochemistry in combination with in situ hybridization, as well as RT-PCR on isolated B lymphocytes, and have determined that the B cells express CB2 mRNA (manuscript submitted for publication). We have also found CB2 mRNA in testis and are currently studying whether there is a spermatogenesis-stage-specific expression of CB2 mRNA. In rat, spermatogenesis consists of 14 stages which can be synchronized with a vitamin A deficient diet. We are beginning to study the levels of CB2 mRNA in the synchronized testis. Thus, we may better understand the role of CB2 in spermatogenesis. The expression of CB1 or CB2 during prenatal development is unknown. We have done in situ hybridization in different aged embryos using probes for CB1 and CB2 mRNA, and found differential expression of each of the receptors as early as day 11 after conception. To study the significance of the presence CB2 in an organism, we will produce transgenic mice lacking the peripheral cannabinoid receptor. We have successfully cloned the mouse CB2 receptor, sequenced the coding sequence, and prepared the plasmid construct for homologous recombination. We have successfully obtained eight clones of embryonic stem cell in which the gene for CB2 was disrupted by homologous recombination. We are now in the process of introducing each of these eight clones into embryos to produce the transgenic mice.